1. Field of the Invention
This invention relates to a polypeptide that has the amino acid sequence of the functional domain of human caldesmon.
2. Description of Related Art
Caldesmon is a protein that can bind with calmodulin, actin and tropomyosin, found in all tissues except skeletal muscle and cardiac muscle. It contributes to the regulation of actomyosin system in smooth muscle. This regulatory function depends on the concentration of calcium ions (flip-flop regulation) in bringing about the regulation of the actomyosin system (Proc. Natl. Acad. Sci. USA, 78, 5652-5655, 1981). When the concentration of calcium ions is low, caldesmon binds with the actin filament-tropomyosin system, and inhibits the mutual interactions of actin and myosin. When the calcium ion concentration is increased, complexes of active calmodulin and caldesmon are formed. This complex is released from the actin filament-tropomyosin system. Because of this, the inhibition of actin-myosin interaction caused by caldesmon cease, and these interactions begin.
Caldesmon was first isolated from the smooth muscles of chicken gizzards, and it has been isolated from other vertebrates. The results of the limited digestion of chicken caldesmons by the protease .alpha.-chymotrypsin have shown that a polypeptide of the size of about 35 kDa at the carboxy-terminal end of chicken caldesmons is the functional unit that brings about flip-flop regulation that depends on the concentration of calcium ions (J. Biochem., 102, 1065-1073, 1987).
There are two isoforms of chicken caldesmon that have different molecular weights. The results of electrophoresis on sodium dodecyl sulfate-polyacrylamide gels have shown that one is of high molecular weight (120,000 to 150,000 Da), now named h-caldesmon, and that the other is of low molecular weight (70,000 to 80,000 Da), now named l-caldesmon. h-caldesmon is abundant in smooth-muscle tissue, and l-caldesmon is abundant in non-muscle tissue and cultured cells. The amino acid sequence of h-caldesmon and the DNA sequence that codes for it have recently been identified (Biochem. Biophys. Res. Commun [called BBRC below], 164,503-511, 1989), as have been the amino acid sequence of 1-caldesmon and the DNA sequence that codes for it (J. Biol. Chem., 266, 355-361, 1991; also Japanese Patent Application 2-37362).
Both h- and l-caldesmons are of chicken origin, and the amino acid sequence of human caldesmon is still unknown.
The morphological change of cells is one characteristic of carcinogenesis. The preservation of cell morphology is closely related to the actin network of the cells, and when cells are transformed, the change in cell morphology occurs in correlation with loss of actin cables, which are part of the filament system that makes up the cytoskeleton (Proc. Natl. Acad. Sci. USA, 72, 994-998, 1975). Immunohistochemical analysis have revealed that caldesmon is localized in actin cables, cell attachment sites, and membrane ruffles in normal cells, but in cancer cells, the compound is not found in any defined cell location (Proc. Natl. Acad. Sci. 81, 3133-3137, 1984).
When cultured cells are transformed with a variety of oncogenic viruses, the caldesmon concentration of the cells decreases and the phosphorylation of the caldesmon increases (Proc. Natl. Acad. Sci. USA 81, 3133-3137, 1984, and Saibo Kogaku [Cell Technology], suppl. 3, "Molecules regulating calcium signalling," 140-153, 1987).